1) Background: Exposure of biologically active materials to ionizing radiation leads to damage to macromolecules and consequent loss of biochemical functions. If the irradiations are carried out at very low temperature, the loss of activities is dependent directly on the molecular mass of the active structures. This procedure - called radiation target analysis - is used to determine the size of the functional unit that carries out important biological activities. These include enzymes, receptors, transporters and others. Often these studies revealed new and unexpected aspects of biomedically important systems. In recent years this laboratory discovered basic differences between proteins and ribonucleic acid in their responses to ionizing radiation. 2) Objective of present studies: a: Fundamental studies of the actions of ionizing radiation on macromolecules are continuing in order to define the exact nature of the damage in different species of molecules. Analyses of these effects by radiation target theory establishes a radiation-sensitive mass associated with measured biological activities. This reveals a fresh perspective in the structure-function relationship in these macromolecules. b: Application of this technique to other enzymes, binding sites, and transporters to determine the size of their active structures, which often is less than the mass of the entire complex. 3) Results during the past year: a: Radiation studies of the hyaluronan synthetase enzyme were conducted both on the native enzyme as well as the same enzyme that had been genetically altered. The gene specifying this enzyme was fused to the gene specifying green fluorescent protein that resulted in production of a single larger protein molecule containing both original proteins. The radiation-sensitive mass increased by an amount equal to the mass of the added protein. This discovery now permits an independent verification and interpretation of target sizes larger than the mass of a single polypeptide. Previous interpretation of such results was that more than one polypeptide was required to perform the measured function, probably a dimer or larger oligomer of the protein. But the results could not exclude the possibility that the additional mass was due to some other protein molecule. Now the genetic fusion technique can be used to resolve this conclusion. If the active structure is a homooligomer, then the genetically-fused protein yields a target size that is increased by the same multiple of mass of the fused protein. However, if the fused protein target size is increased by the mass of only one protein monomer, then the original target size must have included some other proteins. This result therefore extends the capability of radiation target analyses into new areas. b: A previously reported radiation study of an enzyme containing a free-radical, ribonucleotide reductase, showed that under certain conditions the free radical could be removed by radiation products produced elsewhere in the sample. Previously, such results had never been seen. A second free-radical containing enzyme, galactose oxidase, was irradiated to establish the generality of the finding with ribonucleotide reductase. In the oxidase, no inactivation of the free radical was observed unless the radiation interaction was directly in that protein. It was found that radiation could remove free radicals in proteins only if the free radical was accessible to the solvent. 4) Conclusions and significance The technique of radiation target analysis is constantly being tested. Unexpected results have led to further understanding of radiation action directly on macromolecules and extend the applicability of target analysis to new and more complex systems. The results of genetic fusion studies completed this past year now permit a definitive interpretation of the target sizes determined in proteins. A new aspect of radiation effects have been revealed in studies involving free-radical enzymes. Further research in this area will be undertaken to understand the mechanisms involved in this phenomenon.